Project Summary/Abstract There is a well-recognized need for more accurate and cost-effective toxicology screening methods for therapeutic and environmental chemicals. Advances in the field of stem cell-derived brain organoids have led to exploration of these models not only in the study of neural development, but also for drug and toxicity screening. However, because of their cost, complexity, and workflow requirements, these methods have yet to be effectively implemented beyond basic research settings. Such neural organoids are usually formed on a complex extracellular matrix derived from tumors cultured in rodents (commonly known as Matrigel). The process is cumbersome, makes interrogation difficult, and lacks reproducibility. More recently, investigators, including a Stem Pharm co-founder, have demonstrated that brain organoids can be formed, cultured, and assayed reproducibly in a plate-based system on engineered hydrogel substrates with human embryonic stem cell- derived precursor cells. The goal of this project is to apply Stem Pharm?s hydrogel platform to the development and validation of an in vitro mouse neural organoid system for toxicity screening. In specific aim 1, culture conditions will be optimized for the generation of three-dimensional brain organoids derived from mouse embryonic stem cells in 24 well and 96 well plates by adjusting hydrogel characteristics, cell seeding densities, and media replenishment. Reproducibility of the determined method will be assessed via organoid size, neural maturation, and RNA sequencing. Specific aim 2 involves subjecting optimized organoids to a small-scale screen against known developmental neurotoxins. Organoids will be assessed by examining their transcriptome alterations in response to toxin treatments. Finally, the in vitro model will be validated by comparing gene expression patterns to previously generated in vivo toxicant response data. Completion of this study will result in a hydrogel composition and culturing protocols that support the formation of reproducible mouse brain organoids for medium-throughput toxicology screening applications. Phase II studies will utilize data from Phase I transcriptome analyses to identify potential biomarkers, create quantitative molecular panels to assess toxicity, and develop phenotypic screens that will be used to further validate these assays with additional known developmental neurotoxins and non-toxic controls. This work has the potential to drastically reduce the necessity for and costs associated with the use of live animals for toxicity testing. Ultimately, if the responses of these in vitro systems are sufficiently equivalent to or more sensitive than previously generated in vivo animal toxicity data, it would represent critical evidence for the validity of the approach and support the legitimacy of analogous organoid models built with human cells.